Utilizing a cDNA probe specific for bovine cytochrome P-450scc and primary monolayer cultures of bovine adrenocortical cells and human fetal adrenal cells, it has been shown that the chronic action of ACTH on the adrenal cortex includes regulation of P-450scc gene expression at the transcriptional level. The bovine P-450scc cDNA hybridizes strongly to human, pig and rat RNA. Advantage was taken of the cross-reactivity of the bovine P-450scc cDNA with human P-450scc RNA to examine the regulation of P-450scc gene expression by ACTH in human fetal adrenal cells. This process is mediated by cyclic AMP and is inhibited by cycloheximide, in a fashion similar to bovine adrenocortical cells, suggestive that a protein factor(s) activates the response in both species. Hence, the actions of ACTH to regulate P-450scc gene expression in bovine adult adrenocortical cells and human fetal adrenal cells appear to proceed by similar mechanisms.